Chronic Lymphocytic Leukemia

Improving efficiency and sensitivity: European Research Initiative in CLL (ERIC) update on the international harmonised approach for flow cytometric residual disease monitoring in CLL

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Abstract

Detection of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) is becoming increasingly important as treatments improve. An internationally harmonised four-colour (CLR) flow cytometry MRD assay is widely used but has limitations. The aim of this study was to improve MRD analysis by identifying situations where a less time-consuming CD19/CD5/κ/λ analysis would be sufficient for detecting residual CLL, and develop a six-CLR antibody panel that is more efficient for cases requiring full MRD analysis. In 784 samples from CLL patients after treatment, it was possible to determine CD19/CD5/κ/λ thresholds that identified cases with detectable MRD with 100% positive predictive value (PPV). However, CD19/CD5/κ/λ analysis was unsuitable for predicting iwCLL/NCI response status or identifying cases with no detectable MRD. For the latter cases requiring a full MRD assessment, a six-CLR assay was designed comprising CD19/CD5/CD20 with (1) CD3/CD38/CD79b and (2) CD81/CD22/CD43. There was good correlation between four-CLR and six-CLR panels in dilution studies and clinical samples, with 100% concordance for detection of residual disease at the 0.01% (10−4) level (n=59) and good linearity even at the 0.001–0.01% (10−5–10−4) level. A six-CLR panel therefore provides equivalent results to the four-CLR panel but it requires fewer reagents, fewer cells and a much simpler analysis approach.

Introduction

The outcome of chronic lymphocytic leukaemia (CLL) has changed remarkably during the last decades1, 2 with intensive and/or combination therapies now capable of inducing long-lasting clinical remissions in the majority of patients. As many patients achieve complete remissions, the quantification of minimal residual disease (MRD) gained importance. MRD levels during and after therapy were shown to be independent predictors of progression-free and overall survival in CLL.3, 4, 5

Quantification of MRD can be achieved using RQ-ASO IgH-PCR or multi-parameter flow cytometry.6, 7 An international standardised approach for flow cytometric four-CLR analysis has been developed that is applicable to all sample types and treatment regimes.8 However, the procedure is time consuming, may be unnecessary for many patients with obvious CLL present and the interpretation of results may be difficult for non-specialised laboratories.

A simpler flow cytometric approach for MRD detection would include an initial screening for CD19/CD5 coexpression analysis coupled with clonality assessment using surface κ/λ expression.9, 10, 11, 12, 13 This method is not directly quantitative and has variable sensitivity and specificity. However, in some situations the use of highly sensitive MRD flow cytometry may not be necessary, for example, when there are still proportionally high levels of CLL cells in the presence of a normal lymphocyte count. In such situations CD19/CD5/κ/λ alone may be already informative. We therefore aimed at determining whether and when CD19/CD5/κ/λ analysis is sufficient to definitively confirm or exclude the presence of residual disease in situations where a quantitative result is not required. Moreover, with MRD data using CD19/CD5/κ/λ analysis available from recent clinical trials and series, it is important to firmly establish the relationship between those analyses and the quantitative MRD methods.

Conversely, in those cases requiring a comprehensive MRD evaluation, the increasing availability of cytometers that can analyse six or more fluorochromes in parallel makes it possible to reduce the cost and complexity by developing a protocol using six or more CLRs. To this purpose we aimed at developing and standardising a six-CLR antibody panel to reduce sample requirements and time taken for acquisition and analysis.

Patients and methods

Patients

A total of 1155 samples from CLL patients were assessed in this study: 784 samples from patients during or after treatment for comparison of CD19/CD5/κ/λ analysis with four-CLR MRD flow cytometry; 304 samples from patients at presentation or relapse sent for routine diagnostic immunophenotyping (270 for hierarchical cluster analysis of surface antigen expression, 6 samples for dilution studies to investigate the impact of CD3 in different fluorochrome combinations, 22 samples for optimising fluorochromes used and 6 samples for dilution studies to compare four-CLR and six-CLR analysis); and 67 samples from patients with CLL after treatment for comparison of four-CLR and six-CLR MRD flow cytometry. All patients were diagnosed according to the iwCLL/NCI-WG criteria.14 Patients were included before, during and after treatment with chlorambucil, fludarabine-based combinations +/− rituximab, alemtuzumab and autologous/allogeneic transplantation.3, 7, 15, 16, 17, 18, 19 All samples were taken with full-informed patient consent and analysis was performed according to the requirements of the local ethics review committee.

Flow cytometry

Samples were prepared using participating laboratories standard procedures as reported previously, either using ammonium chloride prestaining or FACSlyse post-staining.3, 5, 7, 8, 17, 18, 19 In six CLL cases, dilution studies have been carried on to assess the limit of detection by diluting CLL cells into bone marrow or peripheral blood containing only normal B cells. An initial 1:10 dilution of CLL cells into normal blood/marrow and then six further serial 1:3 dilutions were made to obtain a series of 42 samples with known concentration of CLL cells ranging from 0.001 to 1% of leukocytes.

For evaluation of the requirement to use CD3 to exclude contamination with different antibody combinations, simulated minimal disease samples were prepared by diluting six CLL cases into bone marrow or peripheral blood containing only normal B cells. The cells were incubated with CD19/CD5/CD3 coupled with either CD20/CD79b/CD38, or CD81/CD22/CD43, or CD81/CD79b/CD43. Data for 500 000 events from seven dilutions plus a sample containing only normal B cells were acquired in each case. From each dilution set, six files were generated that were predicted to contain either no CLL events (n=2), 20–50 CLL events (n=2) and 50–100 CLL events (n=2). An identical set of data without CD3 was prepared by electronically removing the information from the original FCS files. The files were blinded and the centres analysed the five-CLR data with no CD3 first, reporting the number of CLL and total B-cell events. Once the participants reported the five-CLR data they were sent the FCS files, which included CD3 data.

The antibody combinations used in the comparison of four-CLR and six-CLR assays are shown in Table 1 and were supplied by BD Biosciences (Erembodegem, Belgium). The same four-CLR antibody combinations were used in the assessment of CD19/CD5/κ/λ analysis with minor exceptions as previously reported.17 A uniform gating strategy for CD19/CD5 clonality assessment was used in all cases, shown in Figure 1. For comparison of CD19/CD5/κ/λ against four-CLR MRD detection and for comparison of four-CLR against six-CLR MRD detection the data was acquired and analysed individually within each laboratory. Analysis of inter-laboratory variation was performed using flow cytometry data files.

Table 1 Antibodies used for four-CLR and six-CLR analysis
Figure 1
figure1

Uniform gating strategy used for evaluation of CD19/CD5/κ/λ expression. (a) Mononuclear cells were identified according to light scatter characteristics (region ‘MNC’). (b) Within the mononuclear cell region, events were subclassified according to CD19 and CD5 expression with vertical and horizontal quadrants set to the edge of the CD19CD5 population. (c) Quadrants for analysis of κ and λ expression were set using the CD19-CD5+ T-cell population to define the lower boundaries for expression of κ or λ light chain, where B cells with light chain fluorescence within the limits of T-cell fluorescence levels were considered to lack expression of that light chain. The quadrants for κ and λ expression were then applied to CD19+CD5 (d) and CD19+CD5+ (e) mononuclear cells. At least 50 events in each region were required to calculate a parameter (for example, to calculate the CD19+ κ:λ ratio would require at least 50 CD19+κ+λ and 50 CD19+κλ+ events). In this case, the B cells represent 1.0% of leukocytes, the CD19+ κ:λ ratio was 1.0:1; 29% of CD19+ cells expressed CD5; the CD19+CD5+ κ:λ ratio was 0.33:1 and 11% of CD19+CD5+ were sIg. The four-CLR MRD analysis detected CLL cells representing 23% of total B cells; the CLL cells had weak sIgλ expression and because of the background of polyclonal CD19+CD5 and CD19+CD5+ B cells, the only parameter to show significant skewing was the CD19+CD5+ κ:λ ratio, although this did not reach a level consistent with 100% PVV for the presence of MRD.

Statistical analysis

The following parameters were calculated from the CD19/CD5/κ/λ tube: (i) the CD19+ and CD19+CD5+ event number and percentage of leukocytes, (ii) the percentage of CD19+ cells expressing CD5, (iii) the percentage of CD19+ and CD19+CD5+ cells lacking surface immunoglobulins and (iv) the κ/λ ratio on CD19+ and CD19+CD5+ cells. For specificity/sensitivity analysis, the four-CLR flow cytometry MRD assay quantitative result was used as the gold standard, with a CLL cell percentage of leukocytes at or above 0.01% (10−4) classified as positive. The PPV was therefore defined as the number of cases above the CD19/CD5/κ/λ threshold for predicting presence of residual disease that had 0.01% (10−4) CLL detected by the four-CLR MRD flow panel (that is, true positive) as a proportion of the total number of cases above the CD19/CD5/κ/λ threshold for predicting presence of residual disease, independent of the four-CLR MRD flow panel result (that is, true positive+false positive).

For evaluation of the requirement to use CD3 to exclude contamination with different antibody combinations (CD19/CD5/CD3 combined with CD20/CD79b/CD38, or CD81/CD22/CD43, or CD81/CD79b/CD43), comparison of the numbers of events classified as CLL from FCS files without CD3 data (electronically removed) against those with CD3 data was performed using a paired t-test analysis.

For assessment of the limit of detection in dilution studies, the error was defined as the difference between observed number of CLL cell events and actual number of CLL cells events as a percentage of actual CLL cell events where the latter was calculated from the known CLL dilution.

Results

CD19/CD5 and clonality assessment for the quick identification of samples with residual disease: a poor negative predictive value

Peripheral blood and bone marrow samples from 784 patients with CLL after treatment were assessed by a basic analysis comprising antibodies to CD19, CD5, κ and λ immunoglobulin light chains and compared with results derived from the complete harmonised four-CLR CLL MRD panel reported previously.8 The assessment of κ and λ ratios were standardised as outlined in Figure 1. CLL cells represented a median 0.5% of leukocytes, range <0.01–23.7%. CLL cells represented <0.01% of leukocytes in 212/784 (27%) of cases, 0.01–1.0% in 206/784 (26%) of cases and more than 1% in 366/784 (47%) of cases.

Clonality assessment is conventionally considered in terms of the ratio of κ+ to λ+ B cells with a ratio below 0.3:1 or above 3:1 typically considered to be abnormal,20 although other thresholds may be more informative.21, 22 The proportion of B cells expressing CD5 under normal circumstances is typically below 30%; in previous studies, threshold levels of 10 (ref. 11) and 25%9, 10 have been used to classify residual disease status but CD5 expression on up to 90% of normal B cells has been reported in regenerating blood after intensive therapy.23 However, MRD can still be detected in a high proportion of cases with a normal B-cell κ:λ ratio and CD5 coexpression levels.24 In this series, the four-CLR MRD analysis detected residual CLL above the 0.01% (10−4) level in 42% (90/213) of individuals with a normal κ:λ ratio (1.0–2.1:1), and in 31% (16/51) of individuals with <10% B-cell CD5 coexpression. Conversely, the four-CLR MRD analysis demonstrated no evidence of residual disease in 5.2% (20/383) of individuals with an abnormal B-cell κ:λ ratio (<0.3 or >3:1) and in 12% (68/545) individuals with more than 30% CD5 expression on the B cells. This datum confirms that the conventional CD19/CD5/κ/λ thresholds have a poor negative predictive value because there is a high proportion of cases with residual disease but normal CD19/CD5/κ/λ levels. However, the positive predictive value (PVV) of CD19/CD5/κ/λ could be informative if it is possible to optimise the thresholds used to identify cases with residual disease.

CD19/CD5 and clonality assessment for the quick identification of samples with residual disease: a 100% positive predictive value

To identify the optimal thresholds for positive prediction of the presence of MRD, the series was divided into a training set and a validation set comprising cases matched for the percentage of B cells and the proportion of cases with 0.01% (10−4) CLL cells by four-CLR (CLR) flow cytometry (training set: n=392, B-cell percentage median 3.93%, range 0.0041–97.9%, 289/392 with 0.01% CLL; validation set: n=392, B-cell percentage median 3.92%, range 0.0038–97.4%, 283/392 with 0.01% CLL).

Specificity and sensitivity analysis was performed on the training set (see Figure 2), and informative thresholds were evaluated on the validation set. As expected, there were no parameters for which a threshold could be identified that showed 100% negative predictive value for the presence of residual disease because a high proportion of cases with normal CD19/CD5/κ/λ have low levels of residual disease. Although a normal CD19/CD5/κ/λ results cannot exclude the presence of residual disease, it was possible to determine threshold levels for parameters within the CD19/CD5/κ/λ analysis that could identify samples with residual disease detectable through the standardised four-CLR assay with 100% PVV. Some of these thresholds (see Table 2) when applied to the validation set were able to confirm a 100% PVV for the presence of residual disease. To obtain such a high PVV, the selected parameters had to be heavily skewed from normal situation. Thus, coexpression of CD5 on more than 82% of B cells, a κ:λ ratio on the CD19+CD5+ B cells 0.05:1 or 32:1 or more than 54% of CD19+CD5+ cells having no detectable surface immunoglobulins were required to predict the presence of MRD with a 100% PPV. Though using these very restrictive criteria, this approach was informative in a high proportion of samples as the presence of residual disease detectable by the four-CLR MRD flow panel could be predicted in 185/392 (47%) samples by simply using CD19/CD5/κ/λ analysis.

Figure 2
figure2

Sensitivity and specificity analysis for identifying residual disease based on the percentage of B cells expressing CD5 in plots (a and b), and the B-cell κ:λ expression ratio in plots (c) and (d). Plots (a and c) show the sensitivity+specificity-1 for identifying the presence of residual disease above a 0.01% (10E-4) threshold by the standardised four-CLR cytometry method, where a value of 1 equates to a threshold with 100% specificity and 100% sensitivity. Plots (b and d) show the specificity for identification of a sample with 0.01% (10E-4) residual disease.

Table 2 Thresholds for predicting the presence of residual CLL using CD19, CD5, κ and λ expression

CD19/CD5 and clonality assessment for the quick identification of samples with residual disease: correlation with clinical response

The correlation between CD19/CD5/κ/λ analysis and iwCLL/NCI response status was evaluated in 27 bone marrow samples from a single centre to determine whether it may be possible to reduce the number of biopsies required for response assessment. Ten of the 27 cases had CD19/CD5/κ/λ parameters within levels associated with a low predictive value for the presence of residual disease (CD19+ <5.5% of total leukocytes, CD19+ κ:λ ratio of 0.5–8.2:1, and in cases with sufficient CD5+ B cells for enumeration <70% of B cells coexpressing CD5, CD19+CD5+ κ:λ ratio of 0.6–5.7:1 and <21% of CD19+CD5+ lacking surface immunoglobulin). Of these cases, 9/10 were in CR and one had morphological evidence of disease (nodular PR). Six of the 27 cases had CD19/CD5/κ/λ parameters above the thresholds indicative of the presence of residual disease but below levels with 100% PVV, of which 3/6 were in CR (1 CRi) and 3/6 were in PR. CD19/CD5/κ/λ parameters were over the thresholds indicative of 100% PVV for detection of residual disease in 11/27 cases (CD19+ >8.9% of total leukocytes, CD19+ κ:λ ratio <0.0.4:1 or >61:1, and in cases with sufficient CD5+ B cells for enumeration >82% of B cells coexpressing CD5, CD19+CD5+ κ:λ ratio of <0.05:1 or >32:1, or >54% of CD19+CD5+ lacking surface immunoglobulin). All 11 of these cases had evidence of residual disease by flow cytometry but only 9/11 had morphological evidence of disease.

CD19/CD5/κ/λ analysis therefore has limited potential to predict the likelihood of morphologically detectable disease in the bone marrow because the analysis may detect very low levels of disease, when normal B cells are absent, and conversely may not detect residual disease, when there is an excess of normal B cells. However, we again confirm that CD19/CD5/κ/λ may be of value in identifying cases that do not require extensive analysis for detection of residual disease.

Development of a six-CLR assay: identifying appropriate marker combinations

CD19/CD5/κ/λ analysis may provide sufficient information in some cases, but in a large proportion of samples a full MRD analysis will still be required. We next investigated the possibility of using six-CLR combinations to minimise the number and complexity of the component tests in the full MRD assays. To identify optimal recombination of the markers present in the four-CLR assay, we first reviewed data on the expression of the key markers in a series of 270 CLL cases (147 peripheral blood, 116 bone marrow and 7 lymph node biopsy samples) to determine whether there were any redundant combinations. Hierarchical cluster analysis was performed with the aim of identifying markers that clustered separately within CLL and may therefore be useful to combine together, but aside from CD20 and CD79b expression which showed a degree of correlation, all the other markers of interest showed independent variation (data not shown).

We next determined whether CD19+CD3+ contaminating events, which cause difficulties in the four-CLR analysis because they have a similar expression profile to CLL cells, could be excluded without requiring the CD3 marker with certain combinations of antibodies. This was achieved by comparing the number of events classified as CLL by operators in three centres analysing files generated by dilution of peripheral blood leuocytes from six patients with CLL diluted into normal leukocytes. The files contained low numbers (<150) of CLL events stained with a combination of CD19/CD5/CD3 with CD20/CD79b/CD38, CD81/CD22/CD43 or CD81/CD79b/CD43. Results from analysis of files with the CD3 data electronically removed were compared against unmanipulated files that were identical apart from the inclusion of CD3 data. The results demonstrate that there is a small though significant reduction in the number of events classified as CLL when CD3 data is included in the gating strategy for a tube containing CD19/CD5/CD20/CD79b/CD38 (n=18, paired t-test P=0.019, regression slope for analysis of data with CD3 vs data without CD3 of 1.13). More interestingly, the inclusion of CD3 becomes relevant particularly for samples with residual disease near the limit of detection using this combination, where it equates to approximately 10–15% reduction in the number of events classified as CLL. In contrast, there was no difference for analyses containing CD19/CD5/CD81/CD22/CD43 or CD19/CD5/CD81/CD79b/CD43, indicating that CD3 should be included in combinations that do not contain CD81/CD43.

Development of a six-CLR assay: optimising fluorochromes

We next tested a different antibody/fluorochrome combinations to determine optimal signal:noise ratio and stability of reagent sets. CD19 and CD5 were included in all combinations with CD79b, CD43, CD20, CD38 tested in different formats in 13 peripheral blood and 9 bone marrow samples (data not shown). It was decided to use CD20 APC-H7 in all combinations for three reasons that are as follows: (i) CD20 as a single marker provides the most powerful separation of CLL cells from normal B cells;24 (ii) combinations that included CD20 did not correlate worse to RQ-PCR than the combination without CD20 in patients treated with rituximab-containing regimens;17 and (iii) most other antibodies available at the time for testing provided a poor signal:noise ratio when conjugated to this fluorochrome. The remaining antibodies in the combinations were chosen to allow the direct or indirect exclusion of contaminating T cells in both tubes. As a result, CD19, CD5, CD20 on PE-Cy7, PerCP-Cy5.5 and APC-H7, respectively, were combined with CD3 FITC, CD38 PE and CD79b APC in the first combination and CD81 FITC, CD22 PE and CD43 APC in the second combination (see Table 1).

FITC, PE, and APC reagents are stable in combination, and PerCP-Cy5.5 stability is not an apparent issue because both PerCP and Cy5.5 emissions are measured by the same detector. However, the stability of tandem conjugates PE-Cy7 and particularly APC-H7 (ref. 25) is less evident and so this was tested over a 28-day period. When all the reagents were combined there was a slight increase in the non-specific APC-H7 signal detected on APC+ cells. This was only detectable in the second combination on T cells with strong CD43-APC binding, which showed a median CD20 APC-H7 fluorescence intensity of 10–20 units for reagents sets prepared at the time of sample preparation increasing to 100–300 units for reagent sets prepared between 1 and 28 days before sample preparation. This change was relatively minor compared with the background fluorescence seen on other cell populations (for example, monocyte non-specific signal of 300–500) and there was no apparent loss of specific PE-Cy7 or APC-H7 signal on B cells. As a result of these differences, a reagent set was prepared comprising two mixtures of the FITC, PE, PerCP-Cy5.5 and APC reagents with CD19 PE-Cy7 and CD20 APC-H7 as separate reagents to be added at the time of sample preparation.

Dilution study analysis

To identify the optimal approach for analysis of both four-CLR and six-CLR data, serial dilutions of peripheral blood leuocytes from six patients with CLL into normal leukocytes were prepared, providing samples with a known level of CLL cells representing a low percentage of total leukocytes (between 0.005 and 1.1%). Although 42 samples were prepared, it was difficult to obtain sufficient events in each of the five four-CLR and both six-CLR data files, and only nine samples were suitable for further analysis. Flow cytometry data files were distributed between participating centres and the files were analysed without knowing the percentage of CLL cells in each file and the results returned for central analysis.

We investigated whether using a minimum number of events required to define a population of CLL cells (no minimum, 20 events, or 50 events) impacted on the coefficient of variation for each dilution and on the percentage error. As all participants analysed the same FCS files, the CV obtained is a measure of the inter-observed variation and does not reflect the variability that might be introduced by repeatedly staining the same aliquot.

As expected, there was a good correlation between four-CLR and six-CLR panels in dilution studies. There was good linearity even below 0.01% (10−4) but the percentage error (defined as the difference between observed number of CLL cell events and actual number of CLL cells events as a percentage of actual CLL cell events) on results was on average greater than 15% for uncorrected data in samples with CLL cells levels between 0.005–0.05%. Assessment of different approaches to control for contamination were assessed by correcting for (that is, subtracting) the numbers of CD19-gated events binding CD3, or by using CD3 as a threshold to determine the limit of detection (that is, only classifying a sample as having detectable residual disease if the number of CLL events is greater than the number of CD19+CD3+ events). The results are shown in Table 3. The most reproducible results with the lowest difference from expected values for both four-CLR and six-CLR analyses were obtained when a minimum of 50 events were utilised to define a CLL population, or when the number of contaminating CD19+CD3+ events was used to define a limit of detection for that sample with a minimum of 20 events utilised to define a population.

Table 3 Assessment of different approaches to reducing error and variation between the observed and expected CLL cell levels in the dilution tests

Comparison of four-CLR and six-CLR assays in clinical samples

A series of 67 samples (54 peripheral blood and 13 bone marrow) were analysed using the harmonised four-CLR MRD assay and the six-CLR assay in parallel, with the aim of testing the reagent set in samples with a low proportion of CLL cells, preferably less than 0.1%. An example of the six-CLR analysis is shown in Figure 3. The median CLL cell percentage reported using the four-CLR assay was 0.008%, and with the six-CLR assay 0.006%. As in the dilution study analysis, the use of a 50 event minimum or the number of CD19+CD3+ events as a threshold for the limit of detection resulted in the best concordance between four-CLR and six-CLR approaches. Overall there was greater than 98.4% concordance, defined as the detection of MRD at either the 0.01% (10−4) level or higher, or below 0.01% (10−4) in both four-CLR and six-CLR assays.

Figure 3
figure3

Example of the approach to analysis of samples for the presence of minimal residual disease using two six-CLR assays. A B-cell gate is defined by the combination of two regions according to CD19 expression and light scatter characteristics (a, b). In the first tube, the number of CD3+ events within the B-cell gate are calculated (c) and the numbers of CLL phenotype events (shown in dark red) are identified by the combination of three regions according to CD5, CD20, CD79b and CD38 expression in (d), (e) and (f). In the second tube, the same B-cell gating approach is applied, and the numbers of CLL phenotype events (shown in dark red) are identified by the combination of three regions according to CD5, CD20, CD22, CD43 and CD81 expression in (g), (h) and (i).

Ideally 500 000 events would be acquired in each test, but because of the difficulties in obtaining sufficient sample to acquire enough events for seven different tubes in post-treatment samples, the reproducibility was limited by a lack of events in some cases. In cases with at least 200 000 events in each data file, (n=59) there was 100% concordance between four-CLR and six-CLR assays in samples. The results are shown in Figure 4. In seven samples (three from Paris where the median number of events captured was over 1 million, and four from Barcelona where at least 500 000 events were captured and the contamination rate was consistently below 20 events), it was possible to compare the detection of CLL phenotype cells in the 0.001–0.01% (10−5–10−4) range by both four-CLR and six-CLR assays. Although the number of samples is small there was good correlation (Pearson R=0.832) with no obvious bias (Bland–Altman limits of agreement 0.0011±0.0028).

Figure 4
figure4

Comparison of four-CLR and six-CLR analysis for the detection of minimal residual disease in samples from patients with CLL after treatment (n=59, of which CLL cells represented 0.001% of leukocytes in 25 cases).

Discussion

The use of MRD analysis in CLL is becoming increasingly common for assessment of response, identifying the kinetics of tumour depletion and relapse, and it is proposed as a potential surrogate end point in clinical trials.5 The harmonised four-CLR assay developed some years ago is highly reproducible and has been validated in multi-centre randomised clinical trials.5, 26 However, in the four-CLR version the assay can be time consuming, particularly in the analysis of minimal levels of disease. In addition the full assay may be unnecessary in samples with higher levels of residual disease.

We have investigated the potential for using the basic CD19/CD5 and clonality assessment to simplify MRD analysis. It was not possible to identify thresholds with a high negative predictive value for the presence of MRD, however, CD19/CD5/κ/λ analysis could be used to identify samples with a very high probability of containing residual disease. In cases with more than 82% of B cells coexpressing CD5, and with a highly skewed κ:λ ratio (<0.05:1 or >32:1) or very weak surface immunoglobulin on the CD5+ B cells (>54% sIg), it was possible to demonstrate in a validation set of 392 cases that there was 100% PVV for the presence of residual disease at the 0.01% (10−4) level or higher. Very large deviations from normal are required for confidently predicting the presence of residual disease for several reasons. First, regenerating B cells have high levels of CD5 expression and even the 82% threshold may be too low after transplantation (SB and NV unpublished observation and Bomberger et al.23). Second, highly skewed κ:λ ratios are frequently seen in the early stages after treatment because the numbers of B cells expressing surface immunoglobulin are low and therefore oligoclonal-reactive expansions can have a major impact on the κ:λ ratio. Lack of surface immunoglobulin expression, (which in this study was defined using T cells as an internal control) on the CD5+ B cells may be most reliable as normal CD5+ B cells should have moderate surface immunoglobulin levels, but the levels of immunoglobulin on CLL cells vary significantly between patients. It is therefore recommended that an excess of CD5+ B cells should only be considered as an indicative of the presence of residual disease if combined with a heavily skewed κ:λ ratio and/or a lack of surface immunoglobulin.

From these results it could be argued that in such cases the use of the full MRD panel would add significant cost and labour time to obtain a quantitative result, when a qualitative evaluation is sufficient in some settings. Therefore, the entire full MRD panel could then be more appropriately restricted to those cases which do not meet the above criteria. Using a CD19/CD5/κ/λ screening approach provided a positive result in 47% of all cases in this series and therefore has the potential to nearly halve the amount of full MRD analyses required in patients receiving standard chemo-immunotherapy protocols. However, CD19/CD5/κ/λ analysis was most effective in samples with higher levels of CLL, predicting the presence of residual disease in 72% (166/232) of cases in the validation set with more than 1% CLL but in only 37% (19/51) of cases with 1% residual CLL. A further limitation of the approach is that it is qualitative only, whereas recent observations clearly demonstrated the added value of MRD quantification within the range of MRD positivity.5, 7

Further time savings may be made by taking advantage of the newer flow cytometry equipment that offers more parameters and the potential to reduce the number of individual assessments required for each case. We have designed a six-CLR assay that incorporates the same markers as the harmonised four-CLR assay but halves the number of tubes required for quantification of the proportion of B-cell which have a CLL phenotype. This can confer modest benefits in terms of the amount of time and cost required to set-up but provides a 50% reduction in the time required to acquire information and provides a major advantages in the time and complexity of analysis, as there are fewer files to analyse in parallel for each case. Also, because the sample is distributed between fewer component tests, the use of a six-CLR analysis permits acquisition of 500 000 events or more even in post-treatment samples, which are often poorly cellular. The results in this study show that when larger numbers of events are analysed, both four-CLR and six-CLR assays can detect CLL cells in the 0.001–0.01% (10−5–10−4) range with good concordance. This places the detection limit of residual disease by flow cytometry in the same level as RQ-ASO IGH-PCR and high-throughput sequencing.17, 27 The ability to reproducibly detect CLL cells at this level would need to be tested in a multi-centre setting, and until such studies are reported it may be preferable to report results of residual disease under the 0.01% (10−4) threshold as being below the known quantitative range. A potential limitation of this study is that the inter-laboratory variation was studied using flow cytometry data files rather than primary samples. However, by analysing the same sample at the same time in multiple laboratories we have previously demonstrated that differences in laboratory preparation do not add significantly to the level of inter-laboratory variation generated by differences in the approach to data analysis.8 The close correlation between laboratories using the four-CLR assay8 coupled with previous reports of detecting residual disease in the 0.001–0.01% (10−5–10−4) range under theoretical conditions28 indicates that quantitative assessment of residual disease below the 0.01% (10−4 CLL cell in 10 000 leukocytes) threshold is achievable.

In summary, we present an approach that can significantly reduce the amount of time and sample required for the enumeration of residual disease in CLL, which can be reliably set-up and run in most diagnostic laboratories with less burden in terms of cost and labour as compared with the current four-CLR flow-cytometric protocol. The use of a simple clonality assessment upfront can also help reducing by around 50% the number of cases that eventually need to go through the full MRD analysis, with additional savings in terms of labour, time and reagents.

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Acknowledgements

We are grateful to BD Biosciences, particularly Frans Nauwelaers and Lucia Testolin, for provision of antibodies used in this study. PG supported by Cariplo Foundation (Milan, Italy), Program Molecular Clinical Oncology-5 per mille number 9965 and Investigator Grant, Associazione Italiana per la Ricerca sul Cancro (AIRC Milano, Italy), Progetti di Ricerca di Interesse Nazionale (PRIN - Ministry of education, University and Research - MIUR, Rome, Italy) and Ricerca Finalizzata 2010 (Ministry of Health, Rome, Italy); AR supported by LLR (www.leukaemialymphomaresearch.org.uk). We are grateful to Linda Falck, Elke Harbst, Jamileh Hanani, Lada Henseleit, Heike Hinrichs and Abdelmalek Dahmani for excellent technical support.

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Correspondence to A C Rawstron.

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Competing interests

The antibodies used for MRD analysis in this study were provided by BD Biosciences. AR has received royalties from BD for an unrelated product (IntraSure). Other companies were contacted at the time of publication of the international standardised approach8 but did not express an interest in providing antibodies for development of MRD detection. BD Biosciences had no scientific input into the project and there were no significant conflicts of interest that could be perceived to bias the work. The remaining authors declare no conflict of interest.

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Rawstron, A., Böttcher, S., Letestu, R. et al. Improving efficiency and sensitivity: European Research Initiative in CLL (ERIC) update on the international harmonised approach for flow cytometric residual disease monitoring in CLL. Leukemia 27, 142–149 (2013) doi:10.1038/leu.2012.216

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Keywords

  • chronic lymphocytic leukaemia
  • minimal residual disease
  • flow cytometry

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